Background: The intestinal epithelium separates us from the microbiota but also interacts with it and thus affects host immune status and physiology. Previous studies investigated microbiota-induced responses in the gut using intact tissues or unfractionated epithelial cells, thereby limiting conclusions about regional differences in the epithelium. Here, we sought to investigate microbiota-induced transcriptional responses in specific fractions of intestinal epithelial cells. To this end, we used microarray analysis of laser capture microdissection (LCM)-harvested ileal and colonic tip and crypt epithelial fractions from germ-free and conventionally raised mice and from mice during the time course of colonization. Results: We found that about 10% of the host’s transcriptome was microbially regulated, mainly including genes annotated with functions in immunity, cell proliferation, and metabolism. The microbial impact on host gene expression was highly site specific, as epithelial responses to the microbiota differed between cell fractions. Specific transcriptional regulators were enriched in each fraction. In general, the gut microbiota induced a more rapid response in the colon than in the ileum. Conclusions: Our study indicates that the microbiota engage different regulatory networks to alter host gene expression in a particular niche. Understanding host-microbiota interactions on a cellular level may facilitate signaling pathways that contribute to health and disease and thus provide new therapeutic strategies.

Skeletal muscle-tissue engineering can be applied to produce cell-based meat for human consumption, but growth parameters need to be optimized for efficient production and similarity to traditional meat. The addition of heme proteins to plant-based meat alternatives was recently shown to increase meat-like flavor and natural color. To evaluate whether heme proteins also have a positive effect on cell-based meat production, bovine muscle satellite cells (BSCs) were grown in the presence of hemoglobin (Hb) or myoglobin (Mb) for up to nine days in a fibrin hydrogel along 3D-printed anchor-point constructs to generate bioartificial muscles (BAMs). The influence of heme proteins on cell proliferation, tissue development, and tissue color was analyzed. We found that the proliferation and metabolic activity of BSCs was significantly increased when Mb was added, while Hb had no, or a slightly negative, effect. Hb and, in particular, Mb application led to a very similar color of BAMs compared to cooked beef, which was not noticeable in groups without added heme proteins. Taken together, these results indicate a potential benefit of adding Mb to cell culture media for increased proliferation and adding Mb or Hb for the coloration of cell-based meat.

Accelerated atherosclerosis diminishes the long term patency of vascular interventions, such as percutaneous coronary intervention and implantation of saphenous vein grafts. However, the cause of this accelerated atherosclerosis is unclear. In this study, we tested the hypothesis that intimal hyperplasia formed following vascular intervention promotes retention of atherogenic lipoproteins. Intimal hyperplasia was surgically induced in the mouse common carotid artery. The surgery was combined with different mouse models of hypercholesterolemia to obtain different cholesterol levels and to control the onsets of hypercholesterolemia. Three weeks after surgery, samples were immunostained for apoB lipoproteins, smooth muscle cells and leukocytes. Already at mild hypercholesterolemia (193 mg/dL), pronounced apoB lipoprotein retention was found in the extracellular matrix in both intimal hyperplasia and the injured underlying media. In contrast, minimal retention was detected in the uninjured proximal region of the same vessel, or in vessels from mice with normal cholesterol levels (81 mg/dL). Induction of aggravated hypercholesterolemia 3 weeks after surgery, when a mature intimal hyperplasia had been formed, caused a very rapid development of atherosclerotic lesions. Mechanistically, we show that lipoprotein retention was almost exclusively dependent on electrostatic interactions to proteoglycan glycosaminoglycans, and the lipoprotein retention to intimal hyperplasia could be inhibited in vivo using glycosaminoglycan‐binding antibodies. Thus, formation of intimal hyperplasia following vascular intervention makes the vessel wall highly susceptible for lipoprotein retention and accelerated atherosclerosis. The increased lipoprotein retention in intimal hyperplasia can be targeted by blocking the interaction between apoB lipoproteins and glycosaminoglycans in the extracellular matrix.

Endothelial expression of tissue-type plasminogen activator (t-PA) is crucial for maintaining an adequate endogenous fibrinolysis. It is unknown how endothelial t-PA expression and fibrinolysis are affected by blood flow in vivo. In this study, we investigated the impact of different blood flow profiles on endothelial t-PA expression and fibrinolysis in the arterial vasculature. Induction of disturbed laminar blood flow (D-flow) in the mouse carotid artery potently reduced endothelial t-PA messenger ribonucleic acid and protein expression, and caused fibrin deposition. En face immunohistochemistry demonstrated that arterial areas naturally exposed to D-flow had markedly lower endothelial t-PA levels than areas with sustained laminar blood flow (S-flow), and displayed pronounced fibrin deposition despite an intact endothelium. In t-PA and plasminogen-deficient mice, fibrin deposition did not extend into S-flow areas, indicating that areas of D-flow and S-flow differ, not only in fibrinolytic capacity, but also in coagulation. Furthermore, plasminogen accumulation was found at D-flow areas, and infusion of recombinant t-PA activated fibrinolysis and significantly reduced the fibrin deposits. In conclusion, D-flow potently impairs the fibrinolytic capacity and causes endothelial fibrin deposition in vivo. Our data also indicate that t-PA is the limiting factor for efficient fibrinolysis at the thrombosis-prone D-flow areas in the arterial vasculature. ^* Per Fogelstrand and Niklas Bergh are senior co-authors of the study.

Background/Aims: Formation of intimal hyperplasia following angioplastic procedures can lead to complications, including restenosis and accelerated atherosclerosis. The vessel wall media is a main source of neointimal cells. However, evidence suggests that there are additional cell sources, such as the adventitia. Here we investigate whether an extensive loss of vascular smooth muscle cells (VSMCs) in the media results in less intimal hyperplasia or if there is compensatory cell recruitment from the adventitia. Methods:A balloon catheter was pulled through the rabbit carotid artery 4 times (major injury) or 2 times (minor injury). Adventitial cells were labeled with 5-bromo-2-deoxyuridine or PKH26. Results:The major injury, but not the minor injury, resulted in a complete loss of VSMCs in large parts of the media and significant leukocyte infiltration. The major injury resulted in less neointima compared with the minor injury. The thinnest neointima was seen at the most injured parts of the media in the major injury group. Cell-tracking experiments showed that the media, but not the adventitia, served as a source of neointimal cells. Conclusion: An augmented angioplastic injury with extensive VSMC loss in rabbits reduced the degree of intimal hyperplasia. No compensatory recruitment of neointimal cells from the adventitia occurred.